ABSTRACT Targeting Hexokinase 2 in Rheumatoid Arthritis (changes are underlined) Hexokinases (HKs) catalyze the first committed step in glucose metabolism. HK2 constitutes the principal inducible isoform and has a restricted distribution of expression in normal adult tissues. Cell populations with increased glycolysis and HK2 expression have a powerful growth advantage. HK2 localizes also at mitochondria, and its interaction increases glucose metabolism and protects mitochondria against apoptosis. Thus, mitochondrial HK2 translocation promotes an activated phenotype in several cell types. Fibroblast like synoviocytes (FLS) and macrophages (MO) are a key component of rheumatoid arthritis (RA) inflamed synovium and contribute to the initiation and perpetuation of destructive joint inflammation. FLS from patients with RA display unique aggressive features, which are autonomous and vertically transmitted. MO are also critical in the pathogenesis of RA. The increase in the number of sublining MO in the synovium is an early hallmark of active rheumatic disease, and high numbers of MO are a prominent feature of inflammatory lesions. Of note, a critical role of glucose metabolism in both activated FLS and MO, have been highlighted by our recent work among others. Our preliminary data demonstrate that while HK1 expression is expressed in both OA and RA synovium, HK2 expression co-localizes with MO and FLS markers, and is only observed in RA and not in OA synovial samples. We also show that HK2 regulates key FLS function as HK2 knockdown impaired FLS invasion. Conversely, HK2 overexpression increases FLS invasion and migration rate. Of note, lactate and PLOD2, which are involved in cell migration and invasion, are upregulated after HK2 expression. Up-regulation of extracellular lactate also suggests a metabolic shift towards accelerated glycolytic metabolism. An HK2 mutant lacking its mitochondrial-binding motif (HK2?N) reversed the invasive phenotype. In MO, a peptide that dissociates HK2 from mitochondria, impaired IL-6 secretion. Importantly, adenovirus-mediated expression of HK2 in the knee by intra-articular injection induced synovial thickness, which was much less evident when HK2?N was intra-articular injected. Finally, HK2F/F-Col1a1 mice, which deletes HK2 in FLS among other non- hematopoietic cells, and treatment with clotrimazole, which dissociated HK2 from mitochondria, significantly decreased arthritis severity. Thus, we will test the hypothesis that mitochondrial HK2 is key regulator of FLS phenotype and MO activation, which contributes to joint destruction in RA. The identification of HK2, an isoform-specific contributor to elevated cell glucose metabolism in RA synovial tissue offers a safer approach than global glycolysis inhibition. HK2 could be selectively targeted without compromising systemic homeostasis or corresponding metabolic function in normal cells as a novel additional approach for combination therapy in RA joint disease independent of systemic immunosuppression.